Xenbase Image ID: 156881

FIG. 7. Region B contains a functional Lef/ -catenin site necessary for positive regulation. A, shown are the results from a mobility shift assay with a fragment of region B including the predicted Lef/ -catenin-binding site as a probe. Purified Lef-1 bound to this fragment (arrow). This binding was inhibited in a dose-dependent manner by adding various amounts (2 to 50) of a DNA fragment including the published Lef-1 consensus binding site. No competition effect was detected with a mutated Lef-1 site (mut. 10 lane). The arrowhead indicates free probe. comp., competitor. B, if Lef-1 was incubated with a GST/ -catenin fusion protein before adding the probe, a DNALef-1 -catenin complex was detected by electrophoretic mobility shift assay (middle arrow). This complex was not very stable under the conditions used, but its existence was confirmed by adding an anti- -catenin antibody (Ab; upper arrow). The lane is a negative control in which the probe was incubated with GST/ -catenin alone. The GST lane is a negative control in which Lef-1 was incubated with GST alone before adding the probe. The arrowhead indicates free probe. C, GFP expression in stage 19 NF embryos injected with the indicated constructs was assessed by RT-PCR. The bars indicate the means S.D. of three independent experiments. The 3000 Lef construct, which contained a promoter lacking the Lef-binding site, gave levels of GFP much lower than those obtained with the full-length 3000 promoter, but similar to those obtained with the negative 70TATA control. a.u., arbitrary units. 30356 Image published in: Vallin J et al. (2001) Copyright © 2001. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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